The abilities of Borrelia burgdorferi, the agent of Lyme disease, to disseminate from the inoculation site at the tick bite and to persist despite the host immune response indicate that interactions with mammalian cells are important continually during infection. B. burgdorferi binds to integrins alphaIIbbeta3 and alphavbeta3, and B. hermsii, an agent of tick-borne relapsing fever, also binds to alphaIIbbeta3, suggesting that integrin binding is important to the arthropod-borne spirochetes. Integrins are key signaling receptors that regulate cell growth, migration, and the inflammatory response. We identified P66, an outer membrane protein of B. burgdorferi, as the ligand for integrins alphaIIbbeta3 and alphavbeta3. Two aspartic acid residues appear to be key to integrin binding by P66, but their roles have not been tested in intact B. burgdorferi. We have also generated B. burgdorferi mutants that do not express P66 and showed that these mutants are defective for beta3 integrin attachment. Since P66 does not contain any classical integrin-binding motifs, the significance of amino acid polymorphisms in P66 from different B. burgdorferi strains with different integrin binding properties, and from different Borrelia species, will provide unique insights into integrin biology and into how the spirochetes affect host cell biology. In Aim 1, the roles of particular P66 amino acids in beta3-chain integrin recognition will be tested by introducing different p66 alleles into our p66-deficient mutant. In Aim 2 we will verify the premise that B. hermsii P66 also binds to beta3-chain integrins, and the importance of amino acid differences between B. burgdorferi and B. hermsii P66 will be explored. In Aim 3 we will investigate how mammalian cell biology is affected by B. burgdorferi P66 attachment to integrin alphavbeta3. Immunofluorescence microscopy, immunoprecipitation, immunoblotting, and microarrays will be used to identify signaling pathways and gene expression changes that are specifically activated by B. burgdorferi P66 binding to alphavbeta3. These experiments will determine how P66-integrin interaction influences the host response to B. burgdorferi. Through this work, we expect to gain insight into how pathogenic spirochetes manipulate the biology of their mammalian hosts, and into how integrins recognize unconventional ligands.